4). Open in a separate window Fig. correlate or surrogate. This study evaluated whether cross-neutralization of HPV types Metroprolol succinate from the Alpha-9 species group is usually mediated by antibodies with a predominantly type-restricted specificity for HPV16 that nevertheless exhibit low affinity interactions with non-vaccine types, Metroprolol succinate or by antibody specificities that demonstrate comparable Metroprolol succinate recognition of vaccine and non-vaccine types but are present at very low levels. Antibodies generated following Cervarix? vaccination of 13C14 12 months old girls were evaluated by pseudovirus neutralization, VLP ELISA and by enrichment of target antigen specificity using VLP-immobilized beads. Two-dimensional hierarchical clustering C1qtnf5 of serology data exhibited that this antibody specificity profile generated by VLP ELISA was both quantitatively and qualitatively different from the neutralizing antibody specificity profile. Target-specific antibody enrichment exhibited that cross-neutralization of non-vaccine types was due to a minority of antibodies rather than by the poor interactions of a predominantly type-restricted HPV16 antibody specificity. Furthermore, cross-neutralization of non-vaccine types appeared to be mediated by multiple antibody specificities, recognizing single and multiple non-vaccine types, and whose specificities were not predictable from examination of the serum neutralizing antibody profile. These data contribute to our understanding of the antibody specificities elicited following HPV vaccination and have potential implications for vaccine-induced cross-protection. strong class=”kwd-title” Keywords: HPV, Vaccine, Antibody 1.?Introduction The human papillomavirus (HPV) vaccines, Cervarix? and Gardasil?, comprise virus-like particles (VLP) based upon the major capsid protein, L1, of HPV16 and HPV18. Both vaccines are highly efficacious at preventing persistent contamination and more progressive disease associated with HPV16 and HPV18 [1,2]. Antibodies capable of neutralizing pseudoviruses representing HPV16 and HPV18 can be detected in the serum and cervicovaginal secretions of vaccinees [3C5]. Together with passive transfer studies demonstrating that immune sera, purified IgG or monoclonal antibodies (MAbs) can protect animals against papillomavirus challenge [6C8], has led to the affordable assumption that vaccine-induced type-specific protection is usually mediated by neutralizing antibodies [9,10]. A degree of cross-protection has also been exhibited against some closely-related types within the Alpha-papillomavirus species groups, Alpha-9 (HPV16-like: HPV31, HPV33, HPV35, HPV52, HPV58) and Alpha-7 (HPV18-like: HPV39, HPV45, HPV59, HPV68) [1,2]. Cross-protection is usually coincident with the detection of cross-neutralizing antibodies against these types in the serum and cervicovaginal secretions of vaccinees [4,11C13]. Whether such antibodies are effectors, or their detection has some power as a correlate or surrogate of vaccine-induced cross-protection is usually uncertain. The antibody response following VLP immunization has been measured using a VLP enzyme-linked immunosorbent assay (ELISA) [14], a pseudovirus-based neutralization assay [15] and a competitive Luminex? immunoassay (cLIA) [16]. Different antibody specificities are measured by each of these assays but the nature of any potential discrepancies are not fully comprehended [9,11]. The cLIA assay uses the type-restricted murine MAb H16.V5 [17], whose human homologue appears to be the majority specificity generated during natural infection [18] and is assumed to constitute a high proportion of the antibodies elicited during vaccination. The magnitude and breadth of the vaccine-induced serum neutralizing antibody response against non-vaccine types generally increases with the vaccine-type response [4,12,13]. It is unclear whether cross-neutralization within the Alpha-9 group is usually facilitated by antibodies other than the H16.V5-like human homologue or that this antibody exhibits some degree of cross-recognition not present in the murine version. In this study we attempted to dissect the serum antibody response generated against non-vaccine types from the Alpha-9 group following Cervarix? vaccination in order to further describe the antibody specificities responsible for cross-neutralization. 2.?Material and methods 2.1. Study samples Serum samples ( em n /em ?=?69) were collected from 13 to 14 year old girls a median 5.9 months following their third dose of Cervarix? [12]. 2.2. L1L2 pseudovirus neutralization assay L1L2 pseudoviruses representing vaccine-relevant Alpha-9 types (HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58) and carrying a luciferase reporter were expressed from transiently transfected 293TT cells, purified and characterized as previously described [12]. The equivalent of a Tissue Culture Infectious Dose 50% (TCID50) was estimated using the SpearmanCKarber equation.